Lecture 13 Electrophoresis (Part-I) · the sample and immobilization of the molecule after...

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Lecture 13 Electrophoresis (Part-I)

Introduction : “Electrophoresis” literally means running in the electric field. The

charged molecule moves to their counter charge electrodes but electric field is

removed before it reaches the electrode. Movement of charged species in an electric

field gives differential mobility to the sample based on the charge and consequently

resolve them. Movement of the charged particle is retarded with the addition a

polymeric gel so that a sufficient time is available for resolving the sample. The

polymeric gel is inert, uncharged and does not cause retardation by binding the

molecule. Instead it, forms pores of different size (depending on the concentration of

polymer) and sample pass through these pore and as a result their electrophoretic

mobility is reduced (Figure 13.1).

Figure 13.1: Movement of the charged particle in an external field.

Suppose a charged particle has net charge Q and the external electric field is E, then

the force F responsible for giving electrophoretic mobility,

F= Q.E…………………………………………….Eq (13.1)

The friction forces F which is opposing the movement of the charged particle is as

follows

F= ƒ. v……………………………………………Eq (13.2),



here ƒ is the friction coefficient and the v is the velocity of the electrophoretic

mobility. The movement of a spherical through a liquid medium (gel) of the viscosity

η, the friction coefficient ƒ is given by :

ƒ = 6Πηrv……………………………………………Eq (13.3)

The place where, F=F or QE=6Πηrv

The electrophoretic mobility v is given by: 𝒗 = 𝐐𝟔𝚷𝛈𝐫

As Q=ze, where z is the valency and e is the electronic charge, the electrophoretic mobility can be expressed as:

𝒗 =𝐳𝐞

𝟔𝚷𝛈𝐫

Hence, electrophoretic mobility v is directly proportional to the charge and inversely

proportional to the viscosity of the medium, size and shape of the molecule. In the

case of relative mobility, it is directly related to the charge/radius of the molecule. For

a globular protein, the radius (r) of the molecule is related to the molecular mass of

the macromolecule. The relative mobility, v’ is as follows

𝒗′ = 𝐂𝐡𝐚𝐫𝐠𝐞𝐦𝐚𝐬𝐬

………………………………………Eq (13.4)

Electrophoretic techniques: Different types of electrophoresis techniques are

designed depending upon whether it carried out in the presence or absence of a

supporting media.

Moving boundary electrophoresis-In this method, the electrophoresis is carried in

solution, without a supporting media. The sample is dissolved the buffer and

molecules move to their respective counter charge electrodes. Moving boundary

electrophoresis is carried out in a U shape tube with platinum electrodes attached to

the end of both arms (Figure 13.2). At the respective ends, tube has refractometer to

measure the change in refractive index of the buffer during electrophoresis due to

presence of molecule. Sample is loaded in the middle of the U tube and then the

apparatus is connected to the external power supply. Charged molecule moves to the

opposite electrode as they passes through the refractometer, a change can be

measured. As the desirable molecule passes, sample can be taken out from the

apparatus along with the buffer.



Figure 13.2: Movement of the charged particle in a moving boundary electrophoresis.

Disadvantages of Moving Boundary electrophoresis-The resolution of the

technique is very low due to the mixing of the sample as well as over-lapping of the

sample components. The electrophoresis technique is not good to separate and

analyze the complex biological sample instead it can be used to study the behavior of

the molecule in an electric field.

Zone electrophoresis-In this method, an inert polymeric supporting media is used

between the electrodes to separate and analyze the sample. The supporting media used

in zone electrophoresis are absorbent paper, gel of starch, agar and polyacrylamide.

The major advantage of presence of supporting media is that it minimizes mixing of

the sample and immobilization of the molecule after electrophoresis. It makes the

analysis and purification of the molecule from the gel much easier than the moving

boundary electrophoresis. The gel electrophoresis is the best example of zone

electrophoresis.



Experiment 13.1 : Analysis of crude cell lysate in SDS-PAGE and determination

of molecular weight of unknown protein.

Principle of the technique: In the discontinuous system, negatively charged

detergent, sodium dodecyl sulphate (SDS), is used to denature protein and impart a

constant negative charge/mass ratio, as a result separation of the protein largely

depends on size only. This technique uses 3 different buffers (1) Running Buffer (2)

Stacking gel buffer of pH 6.8 (3) Resolving gel Buffer of pH 8.8. Discontinuous gel

system concentrate the diluted protein sample into a narrow band, allows application

of diluted protein sample. What is the mechanism of stacking protein sample in

the discontinuous gel system? Stacking gel buffer is composed of TrisHCl pH 6.8,

SDS where as resolving gel buffer contains Tris pH 8.8, SDS and the pore size is

large compared to the resolving gel. The mobility of chloride ion presen in the buffer

is more than the protein in the sample. The glycine moves slower than the protein

sample and as a result protein sample get sandwiched between fast moving chloride

ion and slow moving glycine. Due to high electrophoreitc mobility, the protein

molecules run fast and stack between the leading and trailing ions. Once protein

sample enters into the resolving gel (resolving buffer pH 8.8), glycine ions doesn’t

stop the migration of protein and protein molecules separate as per their size or

molecular weight.

Vertical Gel Instrument- The schematic diagram of a vertical gel electrophoresis

apparatus is given in Figure 13.3. It has two buffer chamber, upper chamber and a

lower chamber. Both chamber are fitted with the platinum electrodes connected to the

external power supply from a power pack which supplies a direct current or DC

voltage. The upper and lower tank filled with the running buffer is connected by the

electrophoresis gel casted in between two glass plates (rectangular and notched).

There are additional accessories needed for casting the polyacrylamide gel such as

comb (to prepare different well), spacer, gel caster etc.



Figure 13.3: Different components of vertical gel electrophoresis apparatus.

Buffer and reagent for electrophoresis- The different buffer and reagents with their

purpose for vertical gel electrophoresis is as follows-

1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide

polymerization.

2. Ammonium persulfate (APS)-it is an initiator for the acrylamide polymerization.

3. Tris-HCl- it is the component of running and gel casting buffer.

4. Glycine- it is the component of running buffer.

5. Bromophenol blue- it is the tracking dye to monitor the progress of gel

electrophoresis.

6. Coomassie brilliant blue R250-it is used to stain the polyacrylamide gel.

7. Sodium dodecyl sulphate-it is used to denature and provde negative charge to the

protein.

8. Acrylamide- monomeric unit used to prepare the gel.

9. Bis-acrylamide- cross linker for polymerization of acrylamide monomer to form

gel.



Casting of the gel: The acrylamide solution (a mixture of monomeric acrylamide and

a bifunctional crosslinker bisacrylamide ) is mixed with the TEMED and APS and

poured in between the glass plate fitted into the gel caster. What is the mechanism of

acrylamide polymerization? Ammoinum persulfate in the presence of TEMED

forms oxygen free radicals and induces the polymerization of acryalide monomer to

form a linear polymer (Figure 13.4). These linear monomers are interconnected by the

cross linking with bis-acrylamide monomer to form a 3-D mesh with pores. The size

of pore is controlled by the concentration of acrylamide and amount of bis-acrylamide

in the gel. In a vertical gel electrophoresis system, we cast two types of gels, stacking

gel and resolving gel. First the resolving gel solution is prepared and poured into the

gel cassette for polymerization. A thin layer of organisc solvent (such as butanol or

isoproponal) is layered to stop the entry of oxygen (oxygen neutralizes the free radical

and slow down the polymerization) and make the top layer smooth. After

polymerization of the resolving gel, a stacking gel is poured and comb is fitted into

the gel for construction of different lanes for the samples (Figure 13.5).



Figure 13.4: Mechanism of acylamide polymerization.



Figure 13.5: Different steps in performace of vertical gel electrophoresis to resolve sample.

Running of the gel: The sample is prepared in the loading dye containing SDS, β-

mercaptoethanol in glycerol to denature the sample and presence of glycerol

facilitates the loading of sample in the well. As the samples are filled vertically there

is a distance drift between the molecules at the top Vs at the bottom in a lane. This

problem is taken care once the sample run through the stacking gel. The pH of the

stacking gel is 6.8 and at this pH, glycine is moving slowly in the front where as Tris-

HCl is moving fast. As a result, the sample gets sandwiched between glycine-Tris and

get stacked in the form of thin band. As the sample enters into the resolving gel with a

pH 8.8, the glycine is now charged, it moves fast and now sample runs as per their

molecular weight (due to SDS they have equal negative charge). After tracking dye

reaches to the bottom of the gel, gel is taken out from the glass plate with the help of a

spatula. Gel is stained with coomassie brilliant blue R250 dye. The dye stains protein

present on the gel. A typical SDS-PAGE pettern is given in the Figure 13.6.



Potentials of discontinuous PAGE :

1. Number of disulfide bonds: Comparison of reducing and non-reducing denaturing gels can be used to provide information related to the number of disulfide bonds present in the protein.

2. Seperating Proteins based on size alone: In the presence of SDS and reducing environment, PAGE gel resolve two proteins of on the basis of molecular masses and the concentration of gel concentration.

In SDS-PAGE, the relative mobility and the log molecular weight as given by

𝒗′ = 𝐕𝐨 𝐀−𝐥𝐨𝐠 𝐌𝐀

………………………………………Eq (13.5)

Molecular weight of a protein can be determined by plotting relative migration Rf with the log molecular weight of standard protein.

𝑹𝒇 = 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐩𝐫𝐨𝐭𝐞𝐢𝐧 𝐟𝐫𝐨𝐦 𝐭𝐡𝐞 𝐥𝐚𝐧𝐞𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐭𝐫𝐚𝐜𝐤𝐢𝐧𝐠 𝐝𝐲𝐞

…………Eq (13.6)

Figure 13.6: Determination of molecular weight using SDS-PAGE. (A) SDS-PAGE (B) Determinaion of Rf.

Calculation of molecular weight of the unknown protein sample is a 5 step process:

1. Resolve the protein sample on the SDS-PAGE along with the molercular weight markers.

2. Calculate the relative mobility (Rf) using the following formula:

𝑹𝒇 = 𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐩𝐫𝐨𝐭𝐞𝐢𝐧 𝐟𝐫𝐨𝐦 𝐭𝐡𝐞 𝐥𝐚𝐧𝐞𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐭𝐫𝐚𝐜𝐤𝐢𝐧𝐠 𝐝𝐲𝐞

…………Eq (13.6)

3. Plot log molecular mass (y-axis) versus relative mobility (x-axis) of the standards.



4. Perform a linear regression using a calculator or using regression software such as Microsoft Excel.

5. Use the linear regression equation (y=mx+c) to estimate the mass of the unknown protein. Log Molecular Weight=(slope)(mobility of the unknown)+Yintercept

Troubleshooting : The pattern of the protein bands on polyacrylamide gel depend on several factors. As a result several types of defects are observed. Now we will discuss few of these defects and responsible factors.

1. Smiling : Uneven heating of the gel causes differential migration of proteins, with outer lanes moving slower than the middle lanes. Rapid heat transfer eliminates this defect and be achived by filling the lower tank with the buffer until sample height.

2. Diffuse Protein Bands: Diffused pattern of the protein band appears on the PAGE. Diffused protein bands pattern can be corrected by increasing running current by 25-50%, higher concentration of acrylamide.

3. Vertical Streaking: Vertical streaking of the protein bands appers due to over-loading of the protein sample. It can be corrected by either reducing amount of the protein sample or running the gel at lower current.

4. Protein Runs faster than expected: In few cases migration of protein is not proportional to the molecular weight, it is either more or less on gel than the expected place. It is due to the unusual very high proportion of basic or acidic amino acids.

5. Double Bands: Appearance of double bands is due to partial oxidation or degradation of the protein sample. Addition of more amount of reducing reagent or preparing a fresh sample will reduce these artifacts.

6. Distorted Protein Bands: Appearance of distorted or uneven protein band is due to stacking gel polymerization. It can be corrected by increasing amount of ammonium persulfate and TEMED or deaerating the stacking gel.

7. Lateral Spreading: if protein bands appears laterally spreading, it can be avoided by reducing the time between loading of the sample and running.



Lecture 14 Staining of PAGE Gels

Other Varients of PAGE:

Native PAGE: SDS-PAGE discussed in the previous lecture is using anionic

detergent sodium dodecyl sulfate and β-mercaptoethanol to give equal charge to all

protein and breaks the disulphide linkage. As a result, the 3-D structure of the protein

is destroyed and it migrate as per their subunit molecular weight. In the native PAGE,

sample is prepared in the loading dye does not contains detergent or denaturating

agent and as a result, sample runs on the basis of charge/mass. In native PAGE, the 3-

D conformation as well as activity of the protein remains unaffected.

Urea PAGE: In this method, insoluable protein is dissolved in Urea and samples

separate based on their charge/subunit mass. A gradient Ura PAGE is used to monitor

the folding states of a protein.

Staining methods in Polyacrylamide Gel: There are different staining protocols are

developed to stain the protein bands in the gel. Few of the selected staining protocol

are given in the Table 14.1 with brief over-view of their advantage and disadvantages.

Table 14.1: Different Stains available for detection of protein in polyacrylamide gels

S.No Type of Stain Features

1 Coomassie Brillian

Blue R250 (CBB)

Non-Specific, One step simple staining procedure,

detection limit 0.3-1µg/protein band

2 Colloidal CBB

Solution

5-10 fold more sensitive than CBB, No destaining step

3 Silver Stain 100 fold more sensitive than CBB, No simple staining

procedure, Mass-spectrometry incompatible

4 SYPRO Ruby Mass-spectrometry compatible, sensitivity upto 1-

2ng/protein band.

5 SYPRO Orange Sensitivity upto 4-8ng/band.

6. SYPRO Tangerine Sensitivity upto 4-8ng/band, can be used to detect

blotted protein bands.



Laboratory Experiment 14.1 : Analyze the crude mixture of protein sample and stain the gel with Coomassie Brilliant Blue R250 dye and perform image analysis. Background Information : Coomassie Brilliant Blue R250 non-specifically stains

almost all proteins and the staining procedure is rapid, simple and inexpensive. The

detection limit of the stain is 0.3 to 1 mg/protein band.

Materials:

1. Polyacrylamide gel containing protein bands.

2. Coomassie Brilliant Blue R250 staining solution:

3. Destaining Solution: 15% (v/v) Methanol and Acetic Acid (10% v/v) in Triple

Distilled water.

4. plastic or glass container with lid.

5. platform shaker

Procedure:

1. Remove polyacrylamide gel from the electrophoresis unit and place in plastic

container with ~10 volumes Coomassie Brilliant Blue R250 staining solution.

2. Agitate slowly on a platform shaker for 30-60mins.

3. Discard staining solution and wash the gel with triple distilled water.

4. Add 5 to 10 gel volumes of destaining solution.

5. Agitate slowly on a platform shaker for 30-60mins.

6. If the color of the destaining solution is intense blue, replace it with the new

destaining solution.

Results: A typical gel picture during coomassie brilliant blue R250 staining and

destaining is given in the Figure 14.1. In the beginning, whole gel will appear

blue/black but the dye will be removed from the background and give discrete

appearance of protein bands.



AFTER STAINING WITH CBB AFTER DE-STAINING

Figure 14.1: Coomassie Brilliant Blue R250 Staining of Polyacrylamide gel.

Image acquisition:

The image acquisition in typical gel documentation has multiple steps as given below:

1. Electrophoresis separation and a suitable staining of the protein bands within the

gel.

2. Preview the gel bands and optimize parameters such as camera focus, brightness

and capture time, magnification, gain, spatial resolution, contrast, gamma etc.

3. Once all parameters are optimized and shows good quality image, image can be

captured.

4. Save the image in a suitable digital image format.

Before discussing image analysis, we will discuss few features of the camera and their

benfits.

1. Number of Pixels : Number of pixels in agiven area increase the spatial resolution

but it comprises a lot on sensitivity and accuracy.

2. Size of Pixels: Smaller pixel size increases the resolution whereas larger pixel size

is more accurate, sensitive and takes lesser time to collect signal.

3. Size of detector: Size of detector decides the field of viewing but it increase the

cost of the optics.



4. Cooling Temperature: Cooling reduces the noise but it is irrelevant for fast

exposures.

5. Readout Noise Specification: lower values can allow to acquire low light

intensity but a slower acquisition may be required to achieve lower readout noise

values.

6. Quantum Efficiency Value: Higher Quantum efficiency is better but it should be

considered along with camera noise.

Analysis of Digital Image: Table 14.2 lists popular imaging and analysis softwares.

Image analysis typically involved several key steps.

STEP 1: Detection of Lane Position: The first step in image analysis is to identify

and mark the lane position. There are different ways to do this taks. For gels with well

defined bands, automatic lane detection algorithm can be able to detect lane. If the

bands are smiling or not straight, then a manual positioning and identifying lane is the

best. There is necessarily consideration while doing manual positioning, the bracket

used should be large enough to cover whole lane but it should not wide to include

bands of other lane.

STEP 2: Detection of Band: Once the lane is defined, protein bands in the lane can

be defined by systematically scanning lane profile and identify the region of local

maxima as band.

STEP 3: Background substraction: Background plays important role in identifying

the protein band as well as measuring the band intensity. Background of a gel picture

is non-uniformaly distributed and made the measurement less accurate. Many

methods of background substration are possible. In one of the method, a replica image

can be generated and then digitally substratec from the original image to correct the

background.



Table 14.2: Different Softwares for 1-D Polyacrylamide Gel Image Analysis

S.No Software Company

1 VisionWorkLS,

DocItLS

UVP Incorporation

2 TotalLab Series Non-Linear Dynamics

3 Gel-Pro, ImagePro MediaCybernetics

4 NIH Image NIH

5 Melanie, Image Master Expasy

6. ImageMaster GE Healthcare

STEP 4: Measurement: Once lane and bands are defined, it is possible to perform

quantification and characterization step. The amount in each band is quantified in

comparison to the background information and the total intensity present in all pixels

present in the band. If the known amount of the protein sample is loaded then a

calibration curve can be drawn and use to more accurately quantitate the band

intensity.

Laboratory Experiment 14.1 : Analyze the crude mixture of protein sample and stain the gel with Colloidal Silver Stain. Background Information : Silver Staining is 50-100 more sensitive than Coomassie

Brilliant Blue R250 stain. But this method is not compatible with the down-stream

mass spectrometry applications.

Materials:

1. Polyacrylamide gel containing resolved protein sample. 2. Fixing Solution A: 40% (v/v) ethanol and 10% (v/v) acetic acid in water. 3. Fixing Solution B: 5% (v/v) ethanol and 5% (v/v) acetic acid in water. 4. 0.5M sodium acetate/1% (v/v) glutaraldehyde 5. 0.05% (w/v) 2,7-napthalene disulphonic acid. 6. Ammonical Silver Nitrate Solution 7. Citric Acid developing Solution 8. Stop Solution: 5% (w/v)Tris base containing 2% (v/v) acetic acid. 9. plastic or glass container with lid. 10. platform shaker



Procedure:

1. Remove PAGE gel from the glass plate and wash with deionized water. 2. remove water and fix the gel in the following manner: A. 1 hr in fixing solution A (10-20 gel volume). B. 2 hr to over-night in fixing solution B (10-20 gel volume). C. wash with deionized water. D. 30mins in 0.5M sodium acetate/1% (v/v) glutaraldehyde (5-10 gel volume) 3. Wash with deionized water. 4. Incubate gel in 5-10 gel volume 0.05% (w/v) 2,7-napthalene disulphonic acid solution for 30mins. 5. Wash with deionized water. 6. Stain gel with 5-10 volume of freshly prepared Ammonical Silver Nitrate Solution. 7. Wash with deionized water. 8. Develop gel with Citric Acid developing Solution. Development will continue until appeartnace of bands. 9. Remove developing solution and stop the reaction with 5% (w/v)Tris base containing 2% (v/v) acetic acid. 10. Wash the gel with deionized water.



Results: A typical SDS-PAGE stained with colloidal silver stain in given in Figure

14.2. Silver staining gives brown colored bands and the number of bands appeared

will be much more than CBB stained gel.

STAINED WITH CBB STAINED WITH SILVER STAIN

Figure 14.2: Comparison of PAGE Gel stained with CBB and colloidal silver stain.



Lecture 15 Western Blotting

Lab Experiment 15.1 : Analyze the E.Coli Over-expressing cells for the

expression of GFP inside the cells.

Background Information: The western blot is a popular technique to detect the

specific protein present in the crude lysate or homogenate. It uses the separation of

different proteins in the gel electrophoresis (preferably SDS-PAGE) and then the

transfer of protein onto a solid support such as nitrocellulose membrane. A primary

antibody direct against protein of interest and then a secondary antibody is used to

detect the primary antibody and gives either colored or chemiluminescent signal.

Figure 15.1: Salient Steps in Western Blotting.

Materials:

1. E.coli cells over-expressing GFP. 2. Protein Standard Markers 3. Transfer Buffer 4. Transfer Membrane: 0.45µm nitrocellulose membrane or PVDF membrane. 5. Plastic Tray 6. Spatula 7. Blotting sheets: 3mm thick cellulose blotting sheets. 8. Semi-Dry Electroblotting Unit. 9. Reagents for performing electrophoresis.



10. anti-GFP antibodies 11. anti rabbit IgG-HRP. 12. Developing reagents Methods : Western Blotting is the multi step process to detect the protein present in

the complex mixture. These Steps are as follows:

STEP 1: Preparation of Sample: Preparation of sample depends on the sample

type.

For Tissue: For solid tissue such as tumor or whole liver, brain, it is first

mechanically broken down into individual cells using a blender, homogenizer or by

sonication. Once individual cells are obtained it will be processed as described.

For Cells: Individual cells are incubated with the lysis buffer containing detergent

along with protease and phosphatase inhibitor cocktail to protect the sample from

degration.

STEP 2: Electrophoresis of Sample: Sample was resolved on SDS-PAGE as

described previously.

STEP 3: Transfer of protein gel on blotting membrane:

A. Preparation of Transfer membrane: Cut the membrane of the same size as gel.

A1: For nitrocellulose membrane: Place the membrane in the transfer buffer and

observe that the liquid has wicked the membrane. Areas appeared as white spot needs

special consideration.

A2: For PVDF membrane: Immerse the membrane into the 100% methanol for 15-

30mins. Decant the methanol and submerge the membrane into the transfer buffer for

additional 10-30mins.

B. Assembly of transfer cassette:

1. Remove the stacking gel from the PAGE and equiliberate the gl in transfer buffer

for 10-30mins.

2. Place a pair of blotting sheet (already saturated with transfer buffer) on the anode

plate (usually this plate is black colored).



3. Place the transfer membrane on the top of blotting sheets and remove trapped air

bubble by rolling test tube or glass rod.

4. Place the PAGE gel on the top of membrane and gently remove trapped air bubble

by rolling test tube or glass rod.

5. Place another blotting sheet (already saturated with transfer buffer) on the top and

remove trapped air bubble by rolling test tube or glass rod.

6. Finally keep cathode plate (usually red colored) and tight the transfer cassette by

four screws. The whole transfer assembly is given in the Figure 15.2.

Figure 15.2: Steps in electroblotting of protein on the membrane.

C. Transfer protein from gel to the membrane: Place the transfer cassette in the

tank filled with the transfer buffer. Connect the transfer apparatus to the power supply

unit and apply constant voltage (100 volts for 1hrs). After transfer, disassembly the

whole cassette and carefully remove transfer membrane and check the protein transfer

by ponseau S stain. Use a pencil and label different lanes.

STEP 4: Blocking: Wash the membrane with distilled water to remove any

remaining ponseau S stain. Put the membrane in blocking buffer containing 5%

skimmed milk (for detection of cytosolic protein) or 5% Fat free BSA (for detection

of phosphor protein) for 30 mins.

STEP 5: Probing: In western blotting, probing can be done in two different ways.

A. Two Step Probing: In two step probing scheme, membrane is first probed with the

primary antibody to recongnize the protein of interest. (Generation of monoclonal or

polyclonal antibody is discussed in the later lectures). Membrane is probed with the

primary antibody with an appropriate dilution for 1hrs at room temp (RT). Membrane

is washed with buffer containing non-anionic detergent Triton X-100 and reprobed



with another antibody directed against the primary antibody. The secondary antibody

is coupled with an enzyme (either HRP or alkaline phosphatase) or a fluorescent dye.

Washed membrane is incubated with the secondary antibody with an appropriate

dilution for 1hrs at room temp (RT). Membrane is washed with buffer containing non-

anionic detergent Triton X-100 and developed. Use of two different antibodies

increases sensitivity as well as giving flexibility to plan multiple probing.

B. One Step Probing: In one step probing, primary antibody contains enzyme or

fluorescent label for detection. One step probing is not very common.

STEP 6: Blot Development : There are multiple ways to develop the blot and detect

the protein present on the membrane. A list of reagents to develop blot are given in

the Table 15.1.

A. Colorometeric Detection: The different colorimetric reagents are given in the

Table 15.1. Wash the membrane with TBS to remove detergent. Place the membrane

into the colorimetric reagent and protein bands appeared in 10-30mins. Stop the

reaction by washing in distilled water. Air dry the membrane and photograph for

permanent record.

B. Chemiluminescent Detection: The different chemiluminescent reagents are given

in the Table 15.1. Transfer the membrane into the chemiluminescent reagent. Soak the

membrane for 30sec to 5mins. Drain off the reagent and wrap the membrane into the

plastic wrap. Place it in film cassette and expose the membrane to film for a few

seconds to several hours.

C. Fluorescent detection: Secondary antibodies labeled with fluorescent dye and

captured in the scanner.



Table 15.1: Different Reagents Available to develop western blot.

System Reagent Reactions

Chromogenic

HRP Based 4-chloro-napthol

DAB/NiCl2

TMB

Oxidized Product to form purple

precipitate

Forms dark brown precipitate

Forms dark purple stain

Alkaline

phosphatase

BCIP/NBT BCIP hydrolysis produces indigo

precipitate after oxidation with NBT.

Luminescent

HRP Based Luminol/H2O2 Oxidized luminal gives off blue light.

Alkaline

Phosphastase

Substituted 1,2 dioxetane

phosphate

Dephosphorylated susbtrate gives off

light



Lecture 16 Electrophoresis (Part-II)

Background Information: The complex biological samples are efficiently resolved

in 2-D gel electrophoresis. It involves the combination of charge and molecular

weight to provide much greater separation in comparison to use of the individual

property. The 2-D electrophoresis is a combination of isoelectrofocusing (IEF)

followed by SDS-PAGE in a perpendicular direction (Figure 16.1). The isoelectric

point (pI) seperates the sample based on their isoelectric pH (it is indirecelty related to

the charge present on the protein) where as SDS-PAGE seperates the molecules based

on sizse (it is indirectly related to the molecular weight). In general analysis of

complex bacterial lysate or tissue extract produces 1000-2500 well separated spots.

With a sensitive detection tool and image analysis software, individual can be

identified and compared under different conditions.

Figure 16.1: An Over-view of 2-Dimensional Gel Electrophoresis. needs redraw from



Lab Experiment 16.1 : Analyze the complex E.coli lysate in 2-D Gel

electrophoresis.

Material and methods:

IPG

Reagents for SDS-PAGE

Reagents for silver stain

Trypsin

MALDI-Tof

Protocol : The complete process of 2-D gel electrophoresis is given in the Figure 16.2 and it has followsing steps:

STEP 1: Protein Extraction

1. Tissue or cells were frozen in liquid and ground to fine powder. The powder was

mixed with chilled (200C) 10% Trichloacetic acid in acetone with 1% DTT.

2. The tissue suspension was incubated for 1hr at 200C and the mixture is centrifuged

at 35000g for 15mins at 40C.

3. Discard the supernatant and carefully dissolve the pellet into the ice cold acetone

containing 1% DTT. Incubate the suspension for 1hr at -200C and subsequently

centrifuge the the mixture at 35000g for 15mins at 40C.

4. Discard the supernatant and pellet was freeze dried multiple times.

5. 25mg of pellet were suspended in 750µl of lysis buffer (9M Urea, 4% CHAPS,

0.8% ampholyte pH 3-10 and 1% DTT) and incubate for 1hr at 370C.

6. Centrifuge the suspension at 12000g at room temperature and collect the

supernatant in fresh tube.

7. Determine the protein concentration by lowry or Bradford method.



Figure 16.2: Different Steps in 2-Dimensional Gel Electrophoresis. needs redraw from

STEP 2: First Dimension Gel Electrophoresis

1. IPG strips (pH 3-10) were rehydrated overnight with 350 µl of rehydration buffer

(8M Urea, 2% CHAPS, DTT (7 mg per 2.5 ml of rehydration buffer) and 0.5% (v/v)

IPG buffer pH 3-10).

2. IPG rehydrated strip loaded with 100µg proteins in a reswelling tray at room

temperature.

3. The focused strips were equilibrated twice (15min each time), Ist equilibration in

50mM Tris-HCl buffer containing 30% (w/v) glycerol, 2% (w/v) SDS, 6M urea, 1%



(w/v) DTT and and 2nd equilibration was performed in a solution containing 4% (w/v)

iodoacetamide instead of DTT.

4. Isoelectric focusing (IEF) was conducted at 20ºC with running conditions as

follows: Ist hr at 500V , followed by 1000V for 2nd hr and finally 16 hrs at 3000V.

The IPG strips will be taken out from the apparatus and 2nd dimensional separation

will be perform by SDS-PAGE in a vertical slab of acrylamide.

STEP 3: Second Dimension SDS-PAGE Electrophoresis

The protocol to perform SDSPAGE is discussed in a earlier lecture.

STEP 4: Gel Silver Staining

The protocol to perform silver staining is discussed in a earlier lecture.



Lecture 17 Horizontal Gel Electrohpresis

Horizontal gel electrophoresis- The electrophoresis in this gel system is performed

in a continuous fashion with both electrodes and gel cassette submersed within the

buffer. The schematic diagram of a vertical gel electrophoresis apparatus is given in

Figure 17.1. The electrophoresis chamber has two platinum electrodes placed on the

both ends are connected to the external power supply from a power pack which

supplies a direct current or DC voltage. The tank filled with the running buffer and the

gel casted is submerged inside the buffer. There are additional accessories needed for

casting the agarose gel such as comb (to prepare different well), spacer, gel caster etc.

Figure 17.1: Different components of horizontal gel electrophoresis apparatus.

Buffer and reagent for electrophoresis- The purpose of each reagents used in

horizontal gel electrophoresis are as follows-

1. Agarose-polymeric sugar used to prepare horizontal gel for DNA analysis.

2. Ethidium bromide- for staining of the agarose gel to visualize the DNA.

3. Sucrose-For preparation of loading dye for horizontal gel.

4. Tris-HCl- The component of the running buffer.

5. Bromophenol blue-Tracking dye to monitor the progress of the electrophoresis.

Casting of the agarose gel- Different steps to cast the agarose gel for horizontal gel

electrophoresis are given in Figure 17.2. The agarose powder is dissolved in a buffer

(TAE or TBE) and heated to melt the agarose. Hot agarose is poured into the gel

cassette and allowed it to set. A comb can be inserted into the hot agarose to cast the



well for loading the sample. In few cases, we can add ethidium bromide within the

gel so that it stains the DNA while electrophoresis.

Figure 17.2: Different steps in casting of the agarose gel for horizontal gel electrophoresis apparatus.

Running and staining-The gel cassette is placed in the electrophoresis tank

submerged completely and DNA loaded into the well with the help of pipetman and

run with a constant voltage. DNA runs from negative to positive end and ethidium

bromide (EtBr) present in the gel stain the DNA. Observing the agarose gel in a UV-

chamber shows the DNA stained with EtBr as organe colored fluorescence (Figure

17.3).

Plasmid DNA

RNA

Figure 17.3: Observation of DNA stained with EtBr in a UV chamber.



Laboratory Experiments related to the Horizontal Agarose Gel Electrophoresis:

Horizontal gel electrophoresis is used to answer several biological, molecular biology

and cell biology question. Here, in today’s lecture we will discuss few selected

example of experimental approaches of horizontal gel electrophoresis-

Lab Experiment 17.1 : Analyze the given DNA sample and determine the

molecular weight using horizontal gel electrohpresis.

Background Information: The size of a DNA can be determined by comparing the size of the known DNA molecules. The DNA of known sizes are resolved on 0.8% agarose along with the unknown sample. Calculation of molecular weight of the unknown DNA sample is a 5 step process:

1. Resolve the DNA on the Agarose Gel along with the DNA molercular weight markers.

2. Calculate the relative mobility (Rf) using the following formula:

Rf=……………………………………………

3. Plot log molecular mass (y-axis) versus relative mobility (x-axis) of the standards.

4. Perform a linear regression using a calculator or using regression software such as Microsoft Excel.

5. Use the linear regression equation (y=mx+c) to estimate the mass of the unknown DNA. Log Molecular Weight=(slope)(mobility of the unknown)+Yintercept

Materials Required: Agarose, Buffers and reagents for horizontal gel as discussed

before.

Procedure: The value of the relative migration (Rf) of each DNA band is calculated

from the agarose gel. The values of relative migration (Rf) and size of the DNA is

used to draw the calibration curve to calculate the size of the unknown DNA samples.



Lab Experiment 17.2 : Analyze the interaction between single stranded binding

protein and different DNA species.

Background Information: DNA is a negatively charged molecule and it interact

with positively charged protein to form DNA-protein complex. The size and the

hydrodynamic volume changes when DNA is interacting with protein to form DNA-

protein complex.

Hence, at the end of the experiment, we can be able to understand several aspects of

DNA-protein interaction:

1. Whether protein-X has a affinity for DNA and the interaction is specific or non-

specific in nature.

2. What will be affinity parameters of the interaction of DNA to protein in making

DNA-protein complex?

Materials Required: Agarose, TAE Buffer, EtBr, Pure SSB, DNA

Procedure: To study the DNA-protein interaction, a fix amount of DNA is incubated

with the increasing concentration of protein (Figure 17.4). Due to the formation of

DNA-protein complex, the hydrodynamic volume of the complex increases and a shift

in band is observed. The DNA has a extended structure and it provides docking site

for several protein molecules such as single stranded binding protein (SSB).

Observation: As a result, a gradual shift in DNA band will be observed until the

DNA binding site is not saturated with the protein molecules.



Figure 17.4: DNA-Protein interaction analysis by agarose gel electrophoresis.

Lab 17.3: Electroelute the given protein sample and determine its concentration.

Background Information- protein band present within the polyacrylamide gel block

is removed by electroelution for further usage (Figure 17.5). During electrophoresis

the protein band migrate and ultimately comes out from the gel block. Due to dialysis

bag, salt and other small molecule contaminant moves out of the dialysis bag but

protein remain trapped within the dialysis bag. Protein can be recovered from the

dialysis bag for further use in downstream processing.

Materials Required: Agarose, Dialysis, Horizontal Gel Tank, Horizontal Tank

Buffer, Dialysis Clips.

Procedure: In this procedure, a gel band is cut from the SDS-PAGE and placed in a

dialysis bag and sealed from both ends. The dialysis bag is choosen so that the

molecular weight cut off of dialysis membrane should be lower than the protein of

interest. The dialysis bag is placed in the horizontal gel apparatus with buffer and

electrophoresis is performed with a constant voltage.

Results and Conclusions: After running the protein isolated from the excised slice

gives protein band in the SDS-PAGE.



Figure 17.5: Electroelution using horizontal gel electrophoresis apparatus.



Lecture 18 RNA gel

Lab Experiment 18.1 : Resolve the mRNA isolated from the liver on the

denaturating Agarose gel electrophoresis.

Background Information: The protocol for RNA gel is similar to the DNA gel

discussed in the previous lecture except that the RNA gels are performed under

denaturating conditions. RNA sample and the agarose gel contains formaldehyde to

denature the secondary structure present in the RNA and prevent the re-formation of

double stranded region in the RNA structure (formation of secondary structure affects

the migration and resolution of RNA on agarose gel). Presence of secondary structure

in RNA allows the faster migration of RNA on agarose gel. As a result, it gives less

time for the molecule to interact with the agarose gel and consequently less resolution

within the different RNA species. Destruction of secondary structures in the RNA

structure minimizes these effects and allows better separation on agarose gel.

Materials:

1. Agarose, Molecular Biology Grade. 2. 10x MOPS Buffer: Composition: 0.4M 3-(N-morpholino)-propanesulphonic acid (MOPS), pH 7.0 0.1M sodium acetate 10mM EDTA, pH 8.0 MOPS buffer can be stored upto 3 months at 40C. For preparing 1xMOPS buffer, it can be diluted with RNase free water. 3. 37% formaldehyde, pH> 4.0 4. RNA molecular weight ladder 5. 0.5M ammonium acetate 6. 0.5µg/ml ehidium bromide in 0.5M ammonium acetate 7. RNase Free H2O 8. Formamide 9. Formaldehyde loading buffer: Composition: 1mM EDTA, pH 8.0 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF 50% Glycerol. Loading buffer can be filter sterilized by passing through 0.22µm filter. It can be aliquot into the small volume and store at -200C. 10. Autoclave gloves 11. 550C water bath



12. Horizontal Gel electrohpresis system 13. Power supply 14. RNase free container for staining and destaining 15. Shaker 16. UV chamber 17. Gel Doc 18. Flask for preparing agarose gel Methods:

1. Isolation of mRNA- The structure of a typical mRNA is given in Figure 18.1. it

has a n-terminal CAP structure, coding sequence and a c-terminal poly A tail. The

Nucleotide A forms 2 hydrogen bonding with nucleotide T and this pairing is very

specific. Exploting this feature, m-RNA population can be isolated from RNA pool

using a poly-T affinity column. The Steps in m-RNA isolation from cell is given in

Figure 18.2. it has following steps:

1. Release of total RNA either by a lysis buffer containing detergent or by

homogenization in the case of hard tissue.

Figure 18.1: Structure of a typical mRNA.

2. Mixing of poly-T containing beads with the toral RNA prepration. Due to mutual

exclusive affinity, mRNA binds to the poly-T beads.

3. Wash the beads with washing buffer to remove non-specific cross contaminating

species.

4. Elute the mRNA from beads and its purity can be checked on polyacryalamide gel

(discussed later in future lectures).



Figure 18.2: Isolation of mRNA from total cell lysate.

2. Preparation and casting of denaturating agarose gel:

(A) Preparation of RNase Free Water:

1. Dissolve diethylpyrocarbonate (DEPC) in deionized distilled water to a final

concentration of 0.1%.

2. Stir this solution for 12hrs at RT and then autoclave to disintegrate DEPC. Store at

RT.

(B) Casting of agarose gel:

1. In a flask, add 1g agarose to the 75ml of RNase free water.

2. Heat the solution to melt the agarose and observe the disappearance of agarose

flecks.

3. Allow the solution to cool down upto 550C. Inside a fuming hood, add 10ml 10x

MOPS buffer and 18ml 37% formaldehyde.

4. Setup the casting tray with comb and pour the gel in fuming hood. [Caution:

formaldehyde is very toxic and can be easily absorbed through skin, wear gloves and

mask].



3. Prepration of RNA sample:

1. Take the RNA sample and make up-to 6.5µl with appropriate quantity.

2. To each sample, add 2.5µl 10x MOPS running buffer, 4.5µl 37% formaldehyde,

11.5µl formamide.

3. Mix by vortexing and briefly spin to collect the sample at the bottom.

4. Inside the hood, add 5µl RNA loading buffer, mix by vortexing and briefly spin to

collect the sample at the bottom.

4. Loading of the RNA sample: Fill the agarose denatured gel prepared with the 1x

MOPS running buffer. Load the RNA sample into the lane (Figure 18.3).

Figure 18.3: Steps in Loading of the RNA samples into the denatured agarose gel.



5. Running the denaturating agarose gel: Place the lid onto the buffer chamber and

perform electrophoresis at 5v/cm until the dye front reaches the 2/3 length of the gel

(Figure 18.4).

Figure 18.4: Running of the denatured agarose gel.

6. Staining of agarose gel:

1. In a RNase-free container, agarose gel is dipped into the 0.5M ammonium acetate

for 40mins at RT.

2. Drain off the solution and dip the block in the 0.5M ammonium acetate containing

0.5µg/ml ethidium bromide.

3. Incubate on RT for 30-40mins.

4. If required and the stain is too intense, it can be destain by 0.5M ammonium acetate

for another 60mins.

5. Transfer the gel to a UV chamber and capture the image with the gel

documentation unit.

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=Uj02zRYHBcV2MM&tbnid=JN9eScgnx410PM:&ved=0CAUQjRw&url=http://biology-forums.com/index.php?topic=21089.0&ei=0ZdwU8XJF4T_rAeJ7oCAAQ&bvm=bv.66330100,d.c2E&psig=AFQjCNFX1dwGDnM6E9_BFhfq_SC_OWn10A&ust=1399974184080162



Lecture 19 Nucleic acid Detection

Lab Experiment 19.1 : Detect the LDH gene in the human genome using

southern blotting.

Introduction: In southern blotting, the genomic DNA is digested with the EcoRI or

BamHI and the DNA fragments are resolved on the agarose gel. The gel is incubated

in an alkaline solution to denature the double stranded DNA to single stranded form.

DNA is transferred on the nitrocellulose membrane by capillary action by applying a

uniform pressure either by suction pressure or by placing wet paper towels. The

membrane is incubated with a non-specific DNA such as sonicated calf thymus

genomic DNA to block the binding sites on the membrane. A single stranded

radioactive probe is added to the membrane and allowed to bind. Membrane is

washed and the blot is developed by autoradiography. The DNA fragment

complementary to the probe sequence binds the radioactive probe and give positive

signal (Figure 19.1).

Figure 19.1: General Over-view of different steps in Southern Blotting.



Materials and Instruments.

Methodology:

A. Restriction Digestion of Genomic DNA:

1. Isolate the high weight molecular weight genomic DNA from the cells.

2. Digest 10-20µg of the genomic DNA with EcoRI (2-3 unit for each µg genomic

DNA) over-night in the presence of appropriate buffer and BSA (if required).

3. Take out small aliquot from the restriction digestion reaction and check on 0.8%

agarose gel. Appearance of smear with visible bands indicate complete digestion of

genomic DNA and the suitability of the sample for southern blotting.

B. Seperation of DNA on agarose gel: Agarose gel electrophoresis is a standard gel

to resolve the DNA and it is discussed in detail in the lecture Lecture 18. As a

standard practice, agarose used for southern blotting is of ultra-pure quality to avoid

containments affecting migration of DNA. Observe the gel in UV-transilluminator

and record the pattern in a gel documentation system.

C. Blotting of DNA on the nylon membrane: Here I am discussing two most

popular methods to transfer the DNA onto the nitrocellulose membrane.

i. Capillary Transfer method: The setup to perform capillary transfer of DNA from

the gel to the nitrocellulose membrane is given in the Figure 19.2.

1. Soak the gel in 0.25N HCl for 10mins with shaking at RT. This step depurinate the

DNA and facilitates the transfer of larger DNA fragments. This setp can be omitted if

the fragmented DNA is of smaller size.

2. Remove the depurination solution. Wash the gel with double distilled water.

Submerge the gel into the alkalike solution for 30mins at RT with gentle shaking.

Alkalike solution will denature the DNA.

3. Remove the denaturation solution. Wash the gel with double distilled water.

Submerge the gel into the neutralization solution for 30mins at RT with gentle

shaking. This step will neutralize gel without denaturing DNA.



4. Pour off the neutrization solution and incubate the gel in the 10xSSC buffer for

30mins with gentle shaking.

5. Preparation of nitrocellulose membrane for transfer: cut a piece of nylon

membrane to the size of agarose gel, mark the nylon membrane by cutting on one

corner. Wash the nylon membrane with the double distilled water and then soak the

membrane in transfer buffer.

6. Cut 2 pieces of 3mm whatman filter paper slightly bigger size to the gel. Wet the

filter paper with the transfer buffer.

7. Setup the blotting tray as given in Figure 20.2. Add 500-800ml transfer buffer

(10xSSC) in the transfer tray. On a glass solid support, place a piece of non-woven

wiper with the size of the solid support to serve as wick. Dip both ends of the wick in

the transfer buffer. Wet the wick and remove the trapped air bubble by rolling glass

rod.

8. Place the gel on the top of wick and remove bubbles by rolling glass rod.

9. Over-lay nylon membrane on the gel and carefully remove the trapped air bubble

by rolling glass rod. (gel and nylon membrane should be handled with wearing

gloves).

10. Carefully lay the filter paper on the membrane, remove the trapped air bubble by

rolling glass rod. Cover the whole setup with saran wrap to avoid loss of untargeted

loss of water.

11. Place a stack of paper towel over the filter paper. Place the glass plate and a

weight (0.5-1kg) on the top of glass plate.

12. Allow the capillary transfer for a period of 4-16 hrs. During this period, if filter

paper gets wet, change the filter to maintain the capillary action. Do not allow the

setup to dry out.

13. Remove the paper towel stack, remove the membrane and rinse with 2xSSC

buffer.

14. Check the DNA transfer blotting on the membrane by visualizing the membrane

in UV light. Under the UV light, mark the positions of DNA and different lanes.



15. Immobilize the DNA on the membrane with a UV cross linker with an optimal

setting.

Figure 19.2: Step up for Capillary Transfer of DNA from gel to the nitrocellulose membrane [Redraw

ii. Vacuum Transfer Methods: Compare to the capillary transfer method, vacuum

transfer method is more efficient. Complete transfer of DNA from gel to the nylon

membrane takes place in couple of hours compared to 16-20hrs in capillary transfer.

Setup of the vacuum blot to transfer the DNA from gel to the membrane is given in

the Figure 19.3.

1. Treat the gel as described in the step 1-5 of the capillary methods.

2. Cut the nylon membrane and the silicon mask with an opening smaller than the

size of the gel.

3. Place the wet 3mm whatman filter paper on the metal grid.

4. Place the nylon membrane on the 3mm whatman filter paper and subsequently put

mask.

5. Position the gel on the mask and place the cover of the vacublot to hold the mask.



6. Apply the vacuum (~75mm Hg) and initiate the transfer process.

7. Pour 5-10ml of depurination solution to cover the gel and let the solution permeate

the gel.

8. Remove the depurination solution and replace it with 20ml denaturation solution

and let the solution permeate through the gel.

9. Replace the denaturation solution with neutralization solution and let the solution

permeate through gel.

10. Replace the neutralization solution with 10x SSC buffer and let the solution

permeate through gel for 30-40mins.

11. Remove excess buffer and turn off the vacuum.

12. Remove the gel and check the transfer under UV.

13. Store the wrapped membrane at room temperature.

Figure 19.3: Step up for vacuum Transfer of DNA from gel to the nitrocellulose membrane



D. Preparation of labeled probe:

1. Preparation of radioactive probe. There are two different method used to label a single stranded DNA probe either at terminal or throught the sequence.

A. Random primer method- In this method, a random primer is used to anneal to the

template and then a PCR reaction is performed in the presence of radiolabeled

nucleotide. After PCR, newly synthesized DNA strand is labeled with radioactive

nucleotide. The whole process is given in Figure 19.4, and it has following steps-

1. The source double stranded DNA is denatured to generate the single stranded DNA

template.

2. A random primer is added and allowed it to anneal to the template strand. It will

anneal to the random position through out the sequence at multiple places.

3. Primer will anneal to the template strand and now klenow will start the synthesis of

new DNA strand.

4. Newly synthesized DNA will give short stretches of multiple labeled DNA probes.

B. Terminal transferease- In this method, a terminal transferase enzyme will label

the probe at the ends to the last nucleotide of the probe. Probe is incubated with the

labeled nucleotide and terminal transferase enzyme will add the labeled nucleotide at

the end. A partial purification with gel filtration column will give labeled primer.



Figure 19.4: Preparation of radioactive probe by random primer method.

E. Hybridization and development of Blot:

1. Place the membrane in a hybridization tube and pre-hybridize in 10-20 ml

hybridization solution at 650C for atleast 30mins.

2. Preparation of probe for hybridization: Denature the probe by boiling for

10mins and quench on ice.

3. Add the probe to the hybridization solution and incubate over-night. Use high

temperature (~650C) for high stringency or moderate temperature (50-550C) for low

stringency conditions.

4. Rinse the membrane thoroughly with 10ml of washing solution 2xSSC containing

0.1% SDS at room temperature.

5. Expose the membrane to an x-ray film with an intensifying screen.

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